Spider venom, a factor that has played a decisive role in the evolution of one of the most successful groups of living organisms, is reviewed. Unique molecular diversity of venom components including substances of variable structure (from simple low molecular weight compounds to large multidomain proteins) with different functions is considered. Special attention is given to the structure, properties, and biosynthesis of toxins of polypeptide nature.

Seven novel short linear antimicrobial and cytolytic peptides named latarcins were purified from the venom of the spider Lachesana tarabaevi. These peptides were found to produce lytic effects on cells of diverse origin (Gram-positive and Gramnegative bacteria, erythrocytes, and yeast) at micromolar concentrations. In addition, five novel peptides that share considerable structural similarity with the purified latarcins were predicted from the L. tarabaevi venom gland expressed sequence tag data base. Latarcins were shown to adopt amphipathic ␣-helical structure in membrane-mimicking environment by CD spectroscopy. Planar lipid bilayer studies indicated that the general mode of action was scaled membrane destabilization at the physiological membrane potential consistent with the "carpet-like" model. Latarcins represent seven new structural groups of lytic peptides and share little homology with other known peptide sequences. For every latarcin, a precursor protein sequence was identified. On the basis of structural features, latarcin precursors were split into three groups: simple precursors with a conventional prepropeptide structure; binary precursors with a typical modular organization; and complex precursors, which were suggested to be cleaved into mature chains of two different types.

This study presents purification, activity characterization, and 1 H NMR study of the novel antifungal peptide EcAMP1 from kernels of barnyard grass Echinochloa crus-galli. The peptide adopts a disulfide-stabilized ␣-helical hairpin structure in aqueous solution and thus represents a novel fold among naturally occurring antimicrobial peptides. Micromolar concentrations of EcAMP1 were shown to inhibit growth of several fungal phytopathogens. Confocal microscopy revealed intensive EcAMP1 binding to the surface of fungal conidia followed by internalization and accumulation in the cytoplasm without disturbance of membrane integrity. Close spatial structure similarity between EcAMP1, the trypsin inhibitor VhTI from seeds of Veronica hederifolia, and some scorpion and cone snail toxins suggests natural elaboration of different functions on a common fold.Antimicrobial peptides (AMPs) 4 are a structurally diverse group of generally small, positively charged peptides produced by various living organisms and demonstrating a wide spectrum of antimicrobial activity (1, 2). Natural sources of AMPs range from prokaryotes to higher animals, and their targets include bacteria, fungi, protozoa, and viruses. The mechanism of action of most known AMPs involves their direct or receptor-mediated interaction with microbial membranes (3-5). It has been generally accepted that membrane-disruptive AMPs kill microorganisms by provoking in different ways an increase in plasma membrane permeability. Non-membrane-disruptive peptides have been shown to target cell wall formation or traverse membranes and affect various internal cellular processes, for example, RNA, DNA, and/or protein biosynthesis. Some AMPs can combine disruptive and non-disruptive mechanisms of action (6). Moreover, mechanisms of action of the same peptide may differ depending on the target. Recent studies have also indicated that AMPs are multifunctional molecules; they can interact with host membrane receptors and influence diverse intracellular processes modulating the immune response of the host organism (7,8).Essential variety in detailed mechanisms of action and multifunctionality imply structural diversity among AMPs. The following structural groups are usually recognized: (i) linear peptides that form ␣-helices in contact with membranes; (ii) disulfide-containing with predominance of ␤-structural elements; and (iii) linear non-␣-helix-forming, usually with a high content of certain amino acid residues (1, 2, 9). Most of the approximately 200 AMP spatial structures known at present (see the Antimicrobial Peptide Database v2.26 (10)) fall into one of the first two groups. Further classification is based on unique features in the sequences and/or structures of AMPs. For example, thionins, defensins, nonspecific lipid transfer proteins, and hevein-and knottin-like peptides have been identified in plants (11-13).To characterize the array of AMPs produced by a plant under certain physiological conditions, we have carried out a systematic analysis of these peptides from...

AMPA receptors mediate fast excitatory neurotransmission and are critical for CNS development and function. Calcium-permeable subsets of AMPA receptors are strongly implicated in acute and chronic neurological disorders. However, despite the clinical importance, the therapeutic landscape for specifically targeting them, and not the calcium-impermeable AMPA receptors, remains largely undeveloped. To address this problem, we used cryo-electron microscopy and electrophysiology to investigate the mechanisms by which small-molecule blockers selectively inhibit ion channel conductance in calcium-permeable AMPA receptors. We determined the structures of calcium-permeable GluA2 AMPA receptor complexes with the auxiliary subunit stargazin bound to channel blockers, including the orb weaver spider toxin AgTx-636, the spider toxin analog NASPM, and the adamantane derivative IEM-1460. Our structures provide insights into the architecture of the blocker binding site and the mechanism of trapping, which are critical for development of small molecules that specifically target calcium-permeable AMPA receptors.

Two forms of a novel antimicrobial peptide (AMP), named WAMP‐1a and WAMP‐1b, that differ by a single C‐terminal amino acid residue and belong to a new structural type of plant AMP were purified from seeds of Triticum kiharae Dorof. et Migusch. Although WAMP‐1a and WAMP‐1b share similarity with hevein‐type peptides, they possess 10 cysteine residues arranged in a unique cysteine motif which is distinct from those described previously for plant AMPs, but is characteristic of the chitin‐binding domains of cereal class I chitinases. An unusual substitution of a serine for a glycine residue in the chitin‐binding domain was detected for the first time in hevein‐like polypeptides. Recombinant WAMP‐1a was successfully produced in Escherichia coli. This is the first case of high‐yield production of a cysteine‐rich plant AMP from a synthetic gene. Assays of recombinant WAMP‐1a activity showed that the peptide possessed high broad‐spectrum inhibitory activity against diverse chitin‐containing and chitin‐free pathogens, with IC50 values in the micromolar range. The discovery of a new type of AMP active against structurally dissimilar microorganisms implies divergent modes of action and discloses the complexity of plant–microbe interactions.

Eight linear cationic peptides with cytolytic and insecticidal activity, designated cyto-insectotoxins (CITs), were identified in Lachesana tarabaevi spider venom. The peptides showed antibiotic activity towards Gram-positive and Gram-negative bacteria at micromolar concentrations as well as toxicity to insects. The primary structures of the toxins were established by direct Edman sequencing in combination with enzymatic and chemical polypeptide degradation and MS. CITs represent a novel class of cytolytic molecules and spider venom toxins. They are the first example of molecules showing equally potent antimicrobial and insecticidal effects. Analysis of L. tarabaevi venom gland expressed sequence tag database revealed the primary structures of the protein precursors; eight peptides homologous with the purified toxins were additionally predicted. CIT precursors share a conventional prepropeptide structure with an acidic prosequence and a processing motif common to most spider toxin precursors. The most abundant peptide, CIT 1a, was chemically synthesized, and its lytic activity on different bacterial strains, human erythrocytes and lymphocytes, insect cells, planar lipid bilayers and lipid vesicles was characterized. The spider L. tarabaevi is suggested to have evolved to rely on a unique set of linear cytolytic toxins, as opposed to the more common disulfide-containing spider neurotoxins.

The multilayered plant immune system relies on rapid recognition of pathogen-associated molecular patterns followed by activation of defenserelated genes, resulting in the reinforcement of plant cell walls and the production of antimicrobial compounds. To suppress plant defense, fungi secrete effectors, including a recently discovered Zn-metalloproteinase from Fusarium verticillioides, named fungalysin Fv-cmp. This proteinase cleaves class IV chitinases, which are plant defense proteins that bind and degrade chitin of fungal cell walls. In this study, we investigated plant responses to such pathogen invasion, and discovered novel inhibitors of fungalysin. We produced several recombinant hevein-like antimicrobial peptides named wheat antimicrobial peptides (WAMPs) containing different amino acids (Ala, Lys, Glu, and Asn) at the nonconserved position 34. An additional Ser at the site of fungalysin proteolysis makes the peptides resistant to the protease. Moreover, an equal molar concentration of WAMP-1b or WAMP-2 to chitinase was sufficient to block the fungalysin activity, keeping the chitinase intact. Thus, WAMPs represent novel protease inhibitors that are active against fungal metalloproteases. According to in vitro antifungal assays WAMPs directly inhibited hyphal elongation, suggesting that fungalysin plays an important role in fungal development. A novel molecular mechanism of dynamic interplay between host defense molecules and fungal virulence factors is suggested.

A new peptide trypsin inhibitor named BWI-2c was obtained from buckwheat (Fagopyrum esculentum) seeds by sequential affinity, ion exchange and reversed-phase chromatography. The peptide was sequenced and found to contain 41 amino acid residues, with four cysteine residues involved in two intramolecular disulfide bonds. Recombinant BWI-2c identical to the natural peptide was produced in Escherichia coli in a form of a cleavable fusion with thioredoxin. The 3D (three-dimensional) structure of the peptide in solution was determined by NMR spectroscopy, revealing two antiparallel α-helices stapled by disulfide bonds. Together with VhTI, a trypsin inhibitor from veronica (Veronica hederifolia), BWI-2c represents a new family of protease inhibitors with an unusual α-helical hairpin fold. The linker sequence between the helices represents the so-called trypsin inhibitory loop responsible for direct binding to the active site of the enzyme that cleaves BWI-2c at the functionally important residue Arg(19). The inhibition constant was determined for BWI-2c against trypsin (1.7×10(-1)0 M), and the peptide was tested on other enzymes, including those from various insect digestive systems, revealing high selectivity to trypsin-like proteases. Structural similarity shared by BWI-2c, VhTI and several other plant defence peptides leads to the acknowledgement of a new widespread family of plant peptides termed α-hairpinins.